Preferentially wrong orientation of inserts in ligation reactions - ive never seen this before... (Sep/01/2009 )

Asc1 and Mlu1 have compatible ends.
I have cut my vector with Asc1 only and dephosphorlyated with antarctic phosphatase.
After cutting my insert with Asc1 and Mlu1 (gel purified) I ligated my vector and insert using T4 DNA ligase. std O/N reaction @ 16*C, 1/3 molar ratio of vector to insert.

Using a control reaction I have determined that 13% of my vectors were ligating to themselves.
So I began by choosing 10 colonies. but all ten of my colonies have been proven by restriction digests and sequencing to be inserted backward.

I re did the ligation but this time at 4*C over the weekend.
I picked 10 colonies again and all of the clones that contained insert were again backward.
subsequently I have picked 10 more colonies and all are backward.

This is very bizarre to me and I hope someone can shed some light and give me some advise.
Thanks and sorry for the long question.

-lumptal-

lumptal on Sep 1 2009, 02:43 PM said:

Asc1 and Mlu1 have compatible ends.
I have cut my vector with Asc1 only and dephosphorlyated with antarctic phosphatase.
After cutting my insert with Asc1 and Mlu1 (gel purified) I ligated my vector and insert using T4 DNA ligase. std O/N reaction @ 16*C, 1/3 molar ratio of vector to insert.

Using a control reaction I have determined that 13% of my vectors were ligating to themselves.
So I began by choosing 10 colonies. but all ten of my colonies have been proven by restriction digests and sequencing to be inserted backward.

I re did the ligation but this time at 4*C over the weekend.
I picked 10 colonies again and all of the clones that contained insert were again backward.
subsequently I have picked 10 more colonies and all are backward.

This is very bizarre to me and I hope someone can shed some light and give me some advise.
Thanks and sorry for the long question.

Hi

This is not unique problem that you are facing. It happens at times. There could be only one reason for it. May be your insert is toxic, that does not allow the bacteria to grow when it is inserted in the right orientation. Try it using a different restriction enzyme or a direction cloning if u have promoters in the gene.

Please post this type of questions under cloning heading where you will get more answers from anyone who has solved the problem.

regards,

jaya

-jaya2020-

it could also be due to DNA secondary structure.

I too have an insert which only ever seems to go in one way, but in this case the fragment goes in with the gene in the correct orientation (lucky me).

Back to your question, toxic gene is a more probable answer, if the gene in question could be expressed in your cell.

You have two options,

f the DNA fragment has a gene with toxic expression in E. coli,
you need to change the promoter to an inducible promoter with tighter control.

if the DNA fragment preferred orientation is due to secondary structure,
use directional cloning. Use two restriction enzymes with incompatible ends. If such enzymes are not naturally available on your DNA insert fragment and vector, you can use primers to introduce your desired restriction sites.